Summary Influenza viruses are the leading cause of human disease due to respiratory viral infection worldwide. It is the overarching objective of this partnership to advance a novel pyrimidine analog anti-influenza virus class towards an investigational new drug-enabling package. The design of this program is driven by our underlying hypothesis that effective next-generation therapeutics for the treatment of influenza must be orally available, display a broad indication spectrum against influenza virus isolates of human, avian, and swine lineages, and covers both influenza A (IAV) and B (IBV) viruses. These product profile demands are derived from the clinical burden imposed by the diverse spectrum of seasonal influenza viruses, the pandemic potential arising from spillover of zoonotic viruses into the human population, and current FDA recommendations that recognize non- hospitalized adults suffering from seasonal influenza as the primary patient population for initial clinical testing. These developmental objectives are best met with direct acting therapeutics, since host-targeted antiviral therapies, although often tantalizingly broad in indication range, are prone to unacceptable side effects that are incompatible with the primary patient group pursued. Under the umbrella of a long-term academia/industry antiviral partnership, we have established a dual- pathogen drug screening protocol that allows the simultaneous automated identification of target virus-specific and broad-spectrum candidates. Implementation of this assay in a large-scale drug screening campaign has yielded a cytidine analog with sub-micromolar antiviral potency. In pilot studies underpinning this preclinical program, we have demonstrated that potent inhibitory activity extends to IAV and IBV isolates, covers viruses representing human and zoonotic lineages, and includes highly pathogenic avian H5N1 and H7N9 viruses of major pandemic threat. The lead compound is orally bioavailable, efficiently converted to the active triphosphate in vivo, and showed sustained micromolar lung tissue concentrations. We have demonstrated oral efficacy in mice against seasonal and highly pathogenic avian influenza viruses with pandemic potential and observed substantial suppression of viral spread in the guinea pig IAV transmission model. In preparation of clinical testing, this lead class will be subjected to mechanistic characterization and resistance profiling (aim 1). In parallel, phospholipid prodrug formulations will be explored to boost drug tissue concentrations for severe disease indications and a structurally independent alternative identified in our screen will be advanced through chemical lead development for back-up to alleviate the potential risk of developmental failure (aim 2). Pharmacokinetic and pharmacodynamic profiles of emerging phospholipid prodrug and back-up leads will be generated and in vivo tolerability determined (aim 3). Efficacy of clinical candidates against seasonal and highly-pathogenic viruses will be tested in mice and ferrets, the effect of prior drug exposure on pathogenesis examined, and the impact on viral spreads assessed in guinea pigs (aim 4).